We propose to study the role of the extracellular matrix in the pathogenesis of hereditary limb malformations in the mouse. We will study embryos with the autosomal dominant gene Dominant hemimelia, Dh, which causes in the hindlimb absence or hypoplasia of the tibia and preaxial polydactyly or oligodactyly associated with anomalies of the femur, ribs and vertebrae. The components of the extracellular matrix to be studied are collagen and glycosaminoglycans (GAG), a logical focus as one human disease causing deformities has been shown to be due to a defect in collagen metabolism and two mutant mice with skeletal anomalies had abnormal GAG's. We will examine initially these substances in two aspects of the developing mutant limb: the mesenchyme-apical ectodermal ridge (AER) interaction and the comparison of the extracellular matrix in the four quadrants (proximal-distal, preaxial-postaxial) of the limb. We will determine whether the mutant is abnormal in the type, amount, timing and sequential appearance of the collagen and GAG's. Studies are done on portions of mouse limbs in short term organ cultures. Collagen production is determined by the incorporation of H3-proline into hydroxyproline, using chromatography to characterize the radioactively labeled collagen. Identification of the type of collagen requires peptide maps produced by cyanogen bromide, molecular sieve chromatography and SDS gel electrophoresis. The GAG's produced are determined by labeling the tissue with H3-acetate and digesting aliquots with 3 different hyaluronidases each of which digests a different GAG. Tissue hyaluronidase activity is determined by the release of N-acetylhexosamine from hyaluronate. Long-term (6 day) organ culture, starting with the limbs of day 11 embryos and going through chondrogenesis, will be used to measure GAG and collagen production, exchange limb components and alter the contents of the media. The findings in Dh/plus embryos will be compared to those in 2 other mutants (luxoid and luxate) with "absent" tibia. The collagen and GAG content in remnants of congenitally "absent" bones of human infants will be measured whenever possible during the 3 yrs.